How to tell if a fish is scared when you aren't a fish yourself

Boy, talk about gaps in the fossil record! It's been nearly a year since my last post. And in any case I didn't work on the touch-me-not plant. I actually worked on signalling in potatoes. Specifically, trying to figure out how the molecule that tells the plant to start forming tubers gets to the tuberization site. Unfortunately, that didn't go very far. I barely started standardizing some experiments and my time was up.

Anyway, new year, new project. I've got bigger fish to fry this time.

Scratch that.

I've got fish to fry. Okay, not fry, but study. This time I'm going to look at how to study anxiety in fish. When we're modelling behavioural responses in other animals, the closer they are to humans, the easier it is to draw direct inferences from observations to human behaviour. However, you can't keep a lab full of monkeys for all your studies for purely practical reasons (and there's the other matter of PETA sloganeering). So people have done a huge amount of work with rats and mice.

Recently though, it's been found that for certain kinds of responses, fish brains are more like humans than mice are. Plus fish are much, much easier to maintain in a lab than mice ever will be, which is a huge advantage.

The thing with behaviour studies is that you need to account for everything. I mean but everything. How regularly the fish are fed, what the temperature of the water is, how much light they get in a day (fish also follow a daily light-dark cycle). And these are only the major, obvious points to look out for. A slight variation in the daily routine can render half your results unusable, so I need to be extra careful (as always!)

Oh, and a friend forwarded me this link. If only teachers here would come up with more interesting assignments!

Mimosa

If there's one thing my blogs lack, it's regularity. This blog, for instance, reads like the fossil record of the earth. Gaps appear and there's no way to guess what's happened in the intervening period. And I don't make any attempt to fill them in. It made me feel guilty at first, but now i'm used to it :)

Anyway, I'm back in college now. Vacation's almost over, which means it's time to get back to the lab. I'm working with a different group this term. This group works on plants. Mostly potato and tobacco, but they're also starting work on the touch-me-not plant. It's suspected that touch-me-not uses many of the same signal molecules as our nerves, which is very cool.

However, until we get to the studying what works how and where, there's a lot of groundwork to be done. For one thing, not many people work on the touch-me-not, so we need to come up with standard, repeatable ways to study everything we study. Otherwise there's no way anybody can verify what we find out. I'll keep you posted as things keep moving.

The weather in Pune is beautiful right now. The roads are wet and muddy, but that's another story. I can see hills outside my window, and they're all covered by a thick carpet of green grass.

It's a good time to be back.

And this is something I found online (sorry, physicists!)

Turning up the heat

I'm on vacation now, and the project reached a... well, not much of a conclusion really. I had to make a presentation right at the end about what I'd done over the last 4 months. And the gist of my presentation was, "It didn't work". You probably saw that coming, what with the cells dying and all

But hey, that's what life in science is about. A successful scientist is one who'd make King Bruce of Scotland look like a quitter of the first order. Nothing you learn in school really prepares you for life in the lab. Come to think of it, nothing you learn in school prepares you for life itself. But that's a topic to be discussed another day.

A couple of days back I was reading this article in The Hindu. And a lot of what it says makes sense. Any bug has to strike a balance between the way it spreads, and how virulent it is. An extremely virulent virus would kill its host before he/she could come into contact with another potential host. Dead host means the virus ain't going anywhere, and consequently the virus is wiped out.

But in a city where you have a few hundred thousand people per square km., it's not difficult to find another host. Especially when we make things easier by squeezing into buses and trains. That's why cities need, absolutely must have, efficient sewerage, water supply and food supply. (Right now, there's an outbreak of cholera in parts of Hyderabad, where I'm currently at. And that just makes the dangers of poor sanitation extremely clear.)

I remember reading about a study where they looked at typhoid outbreaks in South America. They found that the disease was less deadly in countries that had proper sanitation. Apparently this was because good sewerage systems made it difficult for the disease to spread. So it had to hang around in one host longer, to improve its odds of finding another host. And as I said, a bug in a dead host is eventually a dead bug. There was a selection pressure for less virulent bugs, and so less deadly strains evolved. (Score one for the Darwinians!)

The take away? Usual summer advice. Keep yourself hydrated, but try to stick to boiled water. And watch out for cut watermelon sitting in roadside stalls!

They call me the cell killer.

Summer is here, apparently. That’s what one of the PhD students told me. Why? The summer interns have arrived. A boy and a girl. Yes, already. They study in Pune, so they’re getting a head start even though their vacations haven’t started. Plus side? I’m not going to be at the bottom of the lab pecking order now! Both of them seem nice enough. And the girl isn’t hard on the eyes, so that helps :D

It turns out that you can kill Schneider cells (the fly cells) as well. At least it wasn’t a spontaneous mass suicide like those fussy HeLa cells I was working with last semester. Yes, I know for a human derived cell line, HeLa is pretty hardy. But you wouldn’t think so if you could see the kind of abuse S2 can take in its stride and keep going strong. I mean, these cells can survive for 15-20 days with no medium change, nothing. HeLa would’ve died 3 times over by that time.

Anyway, back to the dead S2 cells. It was human error this time. I put the cells in a petri plate in an incubator without a humidifier. Left it there on Friday. Monday morning, the plate’s dry. I mean, properly dry. Why didn’t anybody realize this could happen? We’ve been growing S2 in flasks till now. The flasks have screw-on caps, and S2 cells aren’t too fussy about oxygen concentration. So we screw the caps on tight, and the medium doesn’t evaporate.

The take home message? Petri plates with liquid medium need a humidifier. I’m learning, see?

West side story

About two months back, I’d promised a post about western blots. So here goes.

The principle behind a western blot is the same as electrophoresis. Proteins acquire a negative charge in an appropriate solution. So when a potential gradient is applied, protein molecules will naturally move to the positive electrode.

However, not all proteins are of the same size. So if we place some kind of size-based filter between the electrodes, the smaller molecules would move to the electrode faster. That is the function of the gel in gel electrophoresis. There are pores of a certain size in the gel (the size of the pores can be varied based on the ingredients used) So under a certain potential gradient, with a gel of a certain pore size a protein of certain size/mass can only travel a certain distance in a certain period of time. So a mixture of proteins can be separated on the basis of size by gel electrophoresis. First we stain the proteins before we add them to the gel. That way, we can see where the proteins have been added (A protein solution in water is usually colourless) Alongside the proteins that we study, we also need to add markers. Markers are proteins of a predefined size, which we use like a ruler to gauge the size of the protein we’re studying.

Now that we’ve separated out the mixture of proteins, we need to make sure that the protein we’re looking for is in there. That means detecting with antibodies, since antibodies are extremely specific with the proteins that they bind to. But antibody staining can’t be done in a gel. So the proteins need to be transferred to a durable film of nitrocellulose or polyvinylidene fluoride (PVDF). Once the transfer is complete, antibody staining is done. Staining is a two step process. First, we stain with an antibody that’s specific for the protein we’re interested in. Next we stain with another antibody that’s both specific to the first antibody, and also has fluorescence activity

This is important because it simplifies the procedure. To induce fluorescence activity in an antibody, it needs to be modified artificially. By separating protein recognition from fluorescence, we only need to produce one kind of secondary antibody, which will bind to all primary antibodies regardless of what protein the primary antibody binds to. Instead of building fluorescence into primary antibodies, which means tinkering with pretty much every antibody produced for western blotting.

Why is fluorescence important? It helps us see precisely where our protein is located on the PVDF film and, as a result, on the gel (since the film is like a photocopy of the separation pattern on the gel)

Although I’ve simplified things here, the whole procedure takes a day at least to carry out. Electrophoresis can take anywhere up to 4 hours depending on the protein you’re studying. Transfer of protein onto a PVDF film is done overnight. Primary staining takes three hours, secondary staining takes an hour. And after each staining, the film needs to be rinsed for an hour, otherwise there’s a lot of background fluorescence and you can’t see your protein of interest clearly.

In other news, I’ve shifted lab groups for this semester. My previous guide is on maternity leave till April, so work in her group will go slowly till then. This semester, I’ll still be working on cell cultures. But this time it’ll be fly cell lines, not human. These cells are generally hardier than both HeLa and 293T, so I should have an easier time, in terms of cells dying, over the next few months. Wish me luck!

For want of a nail

Ours is a relatively new lab. Which means it's constantly in the process of being set up. And that means there's new equipment/chemicals/supplies coming in nearly everyday. Right now, my workbench is filled with boxes of pipettes that need to be moved to the store room. And honestly, it isn't comfortable typing with a laptop on your lap. Which kinda defeats the purpose of a laptop, but anyway.

Part of the problem with supplies coming in is that more often than not, somebody messes up somewhere. In the last three months we got reagents we didn't need, equipment we couldn't use and, of course, cells that kept dying on us :) More often than not, it's like that nursery rhyme:

For want of a nail, the shoe was lost;
For want of the shoe, the horse was lost;
For want of the horse, the rider was lost;
For want of the rider, the battle was lost;
For want of the battle, the kingdom was lost;
And all for the want of a horseshoe nail

Case in point, the big-ass centrifuge we had sitting idle for two months in our lab. We couldn't use it because it didn't have the right kind of holders for the tubes we were using. It was pretty frustrating to see that big hunk of metal just sitting there, taking up space.

But that's nothing compared to the trouble we've had getting laminar flow hoods for our cell culturing work. A laminar flow hood is a metal bench which is enclosed from three sides has a door on the fourth and a blower on top. The blower blows finely filtered air through the enclosed space, to prevent contamination by organisms floating in the air. We use two different kinds of hoods for our work. A simpler, more robust kind for working on bacteria (because bacteria are robust), and a more sterile, more expensive kind for animal cell cultures. And right now, we need four of the bacterial ones and two of the animal cell ones. Here's what's happened so far:

Initially, we'd ordered one of the animal cell ones from a local manufacturer. It arrived on time, and did everything we wanted it to do. It wasn't great, but it did the job. We then placed orders for two animal cell ones with an MNC and four bacterial ones with an Indian manufacturer.

The Indian hoods showed up first. And it was pretty messed up. The blower was at the bottom, although the vent was on top, There were holes on the metal workbench, which meant that if we worked with liquid media and if any happened to spill over, it could pour right into the blower mechanism. Plus, the door in front didn't close the way it was supposed to. There was no way we could use these, so we sent them away, and ordered new hoods from another manufacturer.

Next came the MNC hoods, all the way from the U.S. of A. Messed up even more, if anything. It had an annoying alarm that went off if the door was even a millimetre off the "correct" open position. It had no electric sockets (we use mechanical pipettes that need electricity) And even though it was of a different design, the door didn't close properly! Plus, only one showed up. The other hood is still missing.

And today, the hoods we ordered to replace the first bunch of bacterial hoods showed up. Weirdly, these have holes on the back wall, which means your workbench won't be sterile when the blower is off (in case you want to leave your media inside the hood overnight to set, for instance) But the worst part is that they didn't come with legs! That means the workbench is just 6 inches off the ground. Maybe they expect us to work sitting cross-legged on the floor...

And yet, in spite of all this we still manage to get work done. When somebody tells you that life in science isn't easy, believe them!

Go west, young man

I’m back, after a break from the blogosphere, and the lab. I was home for an extended Dusshera-Diwali break. Plus, there wasn’t much happening in the lab over the last month anyway, barring the usual culturing and maintaining of cell stocks. By the way, HeLa has been a bit of a temperamental cell line. I had two sets die on me last week, and that shook me up a little. (My guide says you can never take it easy when it comes to cell culture. Even if you’ve been working with the same cell line for years, decades even, and you know all the ins and outs of your cell line, you can never let your guard down)

 

Anyway, I’m going to run my first western blot today. A western blot is an assay to check for the presence of a specific protein, first by separation based on size and then getting antibodies specific to that protein to bind with it. We’ll know that the antibody has bound to the target protein because it fluoresces on binding. I’m excited because:

a) It’s my first time

b) This is where my project really begins

 

If I haven’t already written about what I’m working on, here goes. My guide is a cancer biologist. She studies the proteins that are activated due to DNA damage. Some of these proteins arrest cell division, till the damage is repaired. It is suspected that these proteins don’t work the way they’re supposed to in cancer cells. We’re trying to confirm that that is what’s happening. Currently, I’m trying to induce DNA damage in HeLa and 293T and then see how expression of these proteins varies from a normal cell from either line. That is, of course, when my cells aren’t dying on me :(

 

I’ll be back later this week with a complete post on how my first western blot went. For now, I’m going to leave you with a bit of trivia. The reason western blotting is called western blotting is because it’s a play on Southern blotting (always a capital ‘S’). Southern blotting was developed by Edwin Southern in 1975 to check for specific DNA. When a technique to quantify protein was developed some time later, they called it western blotting. For RNA, it’s called northern blotting. No, there’s no such thing as an eastern blot, even if it seems unfair. Yes, I know, scientists are a very imaginative lot (rolls eyes)